The principle object of this invention is to provide a new natural extractive principle, enzymatic in type, with fibrinolytic action in vivo, both direct and through plasminogen activation. Since said principle is obtained from the highly vascularized connective tissue of meat animals, it may be named simply and unequivocably `angiokinase` in analogy with urokinase, the proteolytic plasminogen-plasmin activator isolated from the urine of male human subjects (see U.S. Pat. Nos. 2,961,382; 2,983,647; 2,989,440; 3,081,236; see also White et al., Biochemistry, 5, 2160, 1966). Another analogous name is `streptokinase,` another plasminogen activator isolated from hemolytic streptococcus cultures (see U.S. Pat. Nos. 3,016,337; 3,042,586; 3,063,913; 3,063,914; 3,107,203; 3,138,542; 3,276,304).
The significance and the precise meaning of this invention, as well as the utility of its application to the prevention and cure of venal and arterial thrombosis in human subjects will be more readily appreciated after a brief discussion of the currently accepted and widely known view that mammalian blood vessel walls show fibrinolytic action. Said action involves the production of principles specific to this activity, currently known as plasminogen activators. Early views were that the fibrinolytic activity of the vasal wall would predominantly involve the veins and small vessels, but later the activity was observed in large vessels as well, in particular in the wall of the aorta. Research has shown that the blood vessel walls in human subjects killed in accidents (after incanulation of the femoral artery and vein) contained a considerable quantity of so-called `plasminogen activators.`
There was no effective conclusion to the above studies regarding the isolation by chemico-physical extraction of a particular active principle from the vasal wall and from other highly vascularized connective tissues. Therefore, the present invention proposes the isolation and characterization of the active fibrinolytic principle from blood vessels in general and from the aorta in particular, called angiokinase, previously unknown. The invention also relates to its clinical use for therapeutic indications as a general antithrombotic and also within certain limits as a platelet anti-aggregant. In fact, the presence of angiokinase due to the onset of fibrinolytic activity on the part of the walls and then the blood, provides a first level of resistance to thrombosis conditions in warm blooded animals, including humans.
Thus the substance angiokinase, isolated and described in this invention, is the fibrinolytic principle in vasal walls in general and in the aorta in particular, which may be re-added to circulating blood to provide said blood with fibrinolytic potential.
Angiokinase shows the important property that it acts both as an activator (plasminogen plasmin) and as an actual plasmin. This finding is well documented, as described below in support of the invention, and is of considerable theoretical as well as practical significance. Up until now, all studies of the fibrinolytic activity of vasal walls have only revealed the simple activation effects of the pre-formed plasminogen in circulation. Angiokinase however also acts directly in exactly the same way as plasmin.
The areas of use of the present invention will be discussed in detail below.
As is well known, an event of particular importance in circulating pathology, at both venal and arterial levels, is the endovasal deposit of fibrin--said event leads both to stenosis of the vasal lumen and to occlusion (thrombosis).
Fibrin deposition also plays a particularly important role in white platelet thrombus formation, since it is required for adhesion of the platelets to the vasal wall and for platelet aggregation as well as for stabilization of the platelet aggregate.
Keeping the interface between blood and vasal wall free is an important and fundamental process in physiopathology.
It is also well known that the endovasal occlusive event (stenosis and thrombosis) is an indispensable and determining factor for a clinically non-detectable blood vessel condition to become visible on the symptomatic level and to thus become a clinically manifest disease.
This is quite freqnently the case in the athero-arteriosclerotic process which in itself is clinically invisible but becomes evident on the clinical level only (or mainly) when the endovasal stenosis process is accentuated or superceded by actual occlusion.
Therefore, for both prevention and therapy, it is of prime importance to on the one hand block and prevent the formation and accentuation of fibrin deposits and on the other to dissolve these deposits after they are formed.
As is well known, to this end therapy must be instituted to dissolve the fibrin which is forming and the fibrin deposits already formed.
This therapy thus involves the known products with fibrinolytic activity.
The medical profession is in complete agreement regarding the significance and importance of fibrinolytic therapy: it assumes a primary role in circulatory pathology.
There are two conditions underlying fibrinolytic therapy; The first is that, as mentioned above, fibrin is a determining factor in stenosis and endovasal occlusion.
The second is that a deficit in fibrinolytic activity in the blood-vascular system is a pathogenetic factor of prime importance not only in conditioning for the stenosis and vasal occlusion process but also in the same conditioning for atherosclerotic disease.
Too little capacity in the production or release of fibrinolytic activity by the vasal wall is an important factor in determining the atherosclerotic process in general and the endovasal occlusive process in particular. Therefore, a deficit in blood-vasal fibrinolytic capacity has considerable importance for the apthogenesis of atherosclerotic disease as well as for determining its most severe complication--thrombosis.
It is also well known that tissues rich in blood vessels, arterial and venous, are able to produce and release plasminogen activators and thus to show fibrinolytic activity. This then is a tissue activator of plasminogen, meaning the production of fibrinolytic activator at both tissue and vasal levels.
Using the methods of tissue extraction, histo-chemistry and in vitro tissue cultures, tissue and vascular fibrinolytic activity has been demonstrated and widely confirmed, and even measured at natural levels. It is known that tissue activators can be isolated from the heart, lung, ovary, prostate, uterus, etc.: these tissue activators are currently known as TA.
The plasminogen activator, which plays the most important role in intravascular fibrinolysis, is thought to be that synthesized by the endothelial cells topping the blood vessels. Since this enzyme has not yet been isolated and characterized, it has not yet been possible to identify this vascular activity with that of the tissue activator, even though these activities may really be the same.
The vascular or endothelial activator has been the object of numerous studies, and has been recognized and demonstrated, as well as even isolated, from the experimental medicine and biological activity points of view. The plasminogen activator has even been isolated and demonstrated from the vascular tissues of human cadavers.
However, it should be noted that during the thrombotic event, both arterial and venous, the capacity for fibrinolytic production is in deficit and compromised.
Another significant fact to be considered is the reason why this is held to be very important not only for the thrombotic complications arising during the course of ongoing vasculopathies (for example, athero-arteriosclerotic and varicose complications) but also for their pathogenesis, as already mentioned for the presence of a fibrinolytic deficit of the vasal wall.
The lack of plasminogen activators characterized atherosclerotic disease.
A significant correlation has been noted between reduced fibrinolytic activity by the arterial wall and susceptibility to atherosclerosis. A deficity in this activity has been found to be directly proportional to greater and more severe susceptibility and sensitivity to experimentally-induced atheromatous processes.
Moreover, administration of antifibrinolytic agents aggravates the atheromatous process, while administration of fibrinolytic agents attenuates it.
Likewise, administration of antifibrinolytic drugs increases the risk of thrombotic complication, and the risk of systemic thrombotic complications must be taken into account when antifibrinolytic drugs are administered.
On a clinical level, a deficit of fibrinolytic activity has also been shown in atherosclerotic disease, which could be related mainly to a deficit in the production and release of plasminogen activators by the vasal wall and its endothelium in particular.
In atherosclerotic vascular conditions, a real incapacity and reduced power of fibrinolytic response by the vasal wall has been found.
Another particularly important finding has been a deficit of local fibrinolytic activity, or rather of a deficit predominantly corresponding to the vascular regions affected by the atherosclerotic process as compared to less affected and undamaged areas.
With regard to the human myocardium after suffering an infarction (extracts from myocardial tissue soon after an infarction), a sharp reduction of absence of fibrinolytic activity has been found. However, myocardial tissue extracts from infarcted areas in the fibrosis and connective repair phase show said activity.
Analogous results were found in experimentally-induced myocardial infarction: the fibrinolytic activity previously lacking was then found in lesioned areas undergoing repair, because of the plasminogen activators in the newly formed blood vessels.
Another significant finding is that of a fibrinolytic compromission in occlusive venal conditions. Fibrinolytic activity is reduced and compromised in the wall of the thrombus-affected vein. Low fibrinolytic activity in the vasal wall was found to be associated with an increased tendency to develop thrombosis in the deep veins. Thus a large percentage of a large series of patients with severe thromboembolic disease showed decreased fibrinolytic activity of the vasal wall.
The correlation between low fibrinolytic activity of the vasal wall and the blood on the one hand and the onset of thrombosis or pre-thrombotic conditions on the other has been confirmed in several studies.
Diabetes melitis, especially in combination with obesity, has been associated with reduced vascular and/or blood fibrinolysis due to stasis.
It is also known that obesity increases the predisposition of diabetics to vascular occlusions.
With regard to another situation favouring thrombosis development, depressed fibrinolytic activity in the venal wall, and/or reduced release of fibrinolysis activators be the vascular wall, has been found to be a regular post-operatory phenomenon, one that is particularly depressed in those patients who developed thrombosis.
Also, immediately after serious operations, the vascular endothelium cannot respond to stasis as it could before the operation. The term `endothelial decompensation` has been suggested to explain the decrease in fibrinolytic activation in the post-operatory period.
Reduced tissue fibrinolysis is thought to be implicated in post-thrombotic syndrome, with consequent insufficient blood supply to the skin and ulceration.
These clinical and experimental data show that parietal plasminogen activator is greatly decreased in thrombinogenesis, and this observation suggests that these agents are consumed in the attempt to resolve the thrombosis.
Further evidence for the antithrombotic action of the vascular plasminogen activator was obtained when it was found that the greatest danger of experimentally-induced occlusion (for example, during the first eleven days after an operation) generally coincided with reduced or even absent fibrinolytic activity by the vascular wall.
All of the known data in this field have convinced experts in the area that adequate fibrinolytic treatment is an important part of human therapy in the indicated cases.